1. Field of the Invention
This invention generally relates to flow cytometry systems, storage mediums, and methods and further relates to systems, storage mediums, and methods for interrogating and identifying particles flowing through a flow cytometer.
2. Description of the Related Art
Generally, flow cytometers provide measurements of fluorescent intensity of illuminated beads or particles as they pass linearly through a flow chamber. Two or more measurements of fluorescence may be used to classify particles to particular particle subsets. In addition, other fluorescence measurements known as “reporters” may be used to quantify chemical reactions of interest to determine the presence or absence of an analyte in an assay. Each of the fluorescent measurements is made at different wavelengths. In some cases, flow cytometers may further be used to provide measurements of one or more other properties of the particles, such as but not limited to the level of light scattered by a particle and/or the electrical impedance of a particle.
Many conventional flow cytometry measurement systems interrogate particles in two physical locations that are approximately 30 μm-100 μm apart along the direction of fluid flow. At the first interrogation point, a particle is illuminated with its scatter and fluorescence detected simultaneously on three channels, commonly referred to as “DD”, “CL1”, and “CL2”. The same particle is then interrogated at a second point where illumination excites reporter tags that may be bound to the microsphere. This reporter fluorescence is detected on a channel commonly referred to as “RPI”, but other references may be used. In such conventional flow cytometers, particle separation has been estimated to be approximately 400 μm-1,000 μm inside the flow cell. With the separation of particles being many times the distance between interrogation points, there is little chance of two particles being interrogated at two points simultaneously. As such, measurements consecutively collected at the two interrogation points are generally assigned to the same particle. However, as systems and/or techniques are employed where the probability of simultaneous interrogation of multiple particles at different interrogation points increases, accurately correlating pulses generated at different interrogation points within a flow cytometer presents a challenge.